Hormonal Signal Amplification Mediates Environmental Conditions during Development and Controls an Irreversible Commitment to Adulthood

Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it can adopt one of two developmental fates (adulthood or diapause) depending on environmental conditions. The steroid hormone dafachronic acid (DA) directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We used a combination of physiological and molecular biology techniques to demonstrate that commitment to reproductive adult development occurs when DA levels, produced in the neuroendocrine XXX cells, exceed a threshold. Furthermore, imaging and cell ablation experiments demonstrate that the XXX cells act as a source of DA, which, upon commitment to adult development, is amplified and propagated in the epidermis in a DAF-12 dependent manner. This positive feedback loop increases DA levels and drives adult programs in the gonad and epidermis, thus conferring the irreversibility of the decision. We show that the positive feedback loop canalizes development by ensuring that sufficient amounts of DA are dispersed throughout the body and serves as a robust fate-locking mechanism to enforce an organism-wide binary decision, despite noisy and complex environmental cues. These mechanisms are not only relevant to C. elegans but may be extended to other hormonal-based decision-making mechanisms in insects and mammals

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V 267 NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check

A Novel 3-Hydroxysteroid Dehydrogenase That Regulates Reproductive Development and Longevity

A multidisciplinary approach identifies novel biochemical activities involved in the synthesisof C. elegans bile acid-like steroids, which act as hormones that regulate sterol metabolism and longevity

Cooperation of Sumoylated Chromosomal Proteins in rDNA Maintenance

SUMO is a posttranslational modifier that can modulate protein activities, interactions, and localizations. As the GFP-Smt3p fusion protein has a preference for subnucleolar localization, especially when deconjugation is impaired, the nucleolar role of SUMO can be the key to its biological functions. Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants. Upon characterization of sumoylated proteins involved in rDNA maintenance, we established that Top1p and Top2p, which are sumoylated by Siz1p/Siz2p, most likely collaborate with substrates of Mms21p to maintain rDNA integrity. Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p. In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant

Targeting of SUMO substrates to a Cdc48-Ufd1-Npl4 segregase and STUbL pathway in fission yeast

In eukaryotes, the conjugation of proteins to the small ubiquitin-like modifier (SUMO) regulates numerous cellular functions. A proportion of SUMO conjugates are targeted for degradation by SUMO-targeted ubiquitin ligases (STUbLs) and it has been proposed that the ubiquitin-selective chaperone Cdc48/p97-Ufd1-Npl4 facilitates this process. However, the extent to which the two pathways overlap, and how substrates are selected, remains unknown. Here we address these questions in fission yeast through proteome-wide analyses of SUMO modification sites. We identify over a thousand sumoylated lysines in a total of 468 proteins and quantify changes occurring in the SUMO modification status when the STUbL or Ufd1 pathways are compromised by mutations. The data suggest the coordinated processing of several classes of SUMO conjugates, many dynamically associated with centromeres or telomeres. They provide new insights into subnuclear organization and chromosome biology, and, altogether, constitute an extensive resource for the molecular characterization of SUMO function and dynamics

Uncovering Ubiquitin and Ubiquitin-like Signaling Networks

Microscopic imaging and technolog

Disruption of PML Nuclear Bodies Is Mediated by ORF61 SUMO-Interacting Motifs and Required for Varicella-Zoster Virus Pathogenesis in Skin

Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier

SUMO-Interacting Motifs of Human TRIM5α are Important for Antiviral Activity

Human TRIM5α potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5α in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5α shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5α revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5α consensus SUMO conjugation site did not affect the antiviral activity of TRIM5α in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5α antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5α restriction of N-MLV. Our data suggest a novel aspect of TRIM5α-mediated restriction, in which the presence of intact SIMs in TRIM5α, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA

Identification of sumoylation targets, combined with inactivation of SMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogen Candida albicans

Peer reviewedPublisher PD